Peptide and uses therefor

ABSTRACT

Provided is a method for treatment and/or prophylaxis of a condition associated with T cell mediated chronic inflammatory disease by administration, to a patient, of a peptide comprising N′-SVTEQGAELSNEER-C′ (SEQ ID NO: 1) or an analogue thereof that inhibits T cell migration. Also provided is the peptide or its analogue for use in the methods of treatment and/or prophylaxis of said condition. Also provided is a method for the treatment of Sjogren&#39;s syndrome by administration of a peptide comprising N′-SVTEQGAELSNEER-C′ to a patient in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.14/370,881, filed on Jul. 7, 2014, which was the national stage ofInternational Application No. PCT/GB2013/050068, filed on Jul. 14, 2013,all of which are incorporated by reference herein.

REFERENCE TO SEQUENCE LISTING

A Sequence Listing is submitted electronically via EFS-Web as an ASCIIformatted text file with the name “7492123C1SeqList”; the file wascreated on Nov. 16, 2016, is 16 kilobytes in size, and is incorporatedherein by reference in its entirety.

BACKGROUND

Field of Invention

The present invention relates to use of a peptide secreted from B cellsthat has an inhibitory effect on the migration of T cells (includingauto-reactive T cells). This has applications in the treatment and/orprophylaxis of the conditions associated with such T cells, most notablytype 1 diabetes mellitus and Sjogren's syndrome.

Related Art

Pancreatic islet-reactive T cells play a central role in beta celldestruction and thus in the pathogenesis of type 1 diabetes (T1D). Inevidence, T cells comprise a major part of the islet infiltrate in a T1Dpancreas, and immunosuppressive drugs that target T cells preserve betacell function. Understanding the mechanisms by which islet-reactive Tcells are recruited from the blood, across inflamed endothelium, andinto the pancreatic islet have been poorly examined in T1D.

This is particularly relevant because healthy humans can also havecirculating islet reactive T cells that do no apparent harm. Therefore,we believe that in T1D, endogenous mechanisms that prohibit thetrafficking of reactive T cells into the pancreas fail, and if suchregulatory pathways could be re-established it may be possible toexclude auto-reactive T cells and preserve beta cell function. Theadipocyte-derived cytokine, adiponectin, has a role to play inregulating T cell migration, but the picture is more complex than thatas adiponectin's circulating levels do not seem to fluctuate in T1D.

T cells also play a central role in the pathology of the chronicinflammatory autoimmune disease primary Sjogren's syndrome (pSS).Sjogren's syndrome is characterized by loss of function of secretoryglands, for example salivary or tear glands and autoantibody production.Lymphocyte infiltration of the salivary glands and formation oforganised inflammatory aggregates of T and B-lymphocytes represent thehistological hallmark of the disease. The formation of these aggregates,often organised in lymph node-like structures with formation offunctional germinal centers, is associated with worsening diseaseprognosis, higher production of autoantibodies and development of extranodal mucosa-associated lymphoid tissue (MALT) lymphoma. To date thereis no approved therapy. In addition there are at present no diagnostictools for early identification of pSS or to stratify patients that wouldbenefit from immunosuppressive treatment. Thus, pSS is considered anorphan disease.

SUMMARY

Surprisingly, we have found that a certain peptide can inhibit T cellmigration. Although this peptide is known, what we have shown is thatadiponectin achieves its effects on T cell migration by the induction ofa mediator released from B lymphocytes. This mediator, a peptide,appears to be an inhibitor of T cell trans-endothelial migration.

The peptide has the sequence N′-SVTEQGAELSNEER-C′ or is an analoguethereof that inhibits T lymphocyte migration.

Thus, in a first aspect, the present invention provides a method fortreatment and/or prophylaxis of a condition associated with T cellmediated chronic inflammatory disease by administration of a peptidecomprising N′-SVTEQGAELSNEER-C′ to a patient in need thereof. Thepeptide may also be an analogue or variant thereof that inhibits Tlymphocyte migration.

The condition is, optionally, selected from the group consisting of Tcell auto-reactivity, T cell mediated chronic inflammatory disease andautoimmune disease. Alternatively, the condition may be T cellauto-reactivity or T cell mediated chronic inflammatory disease orautoimmune disease.

It will be appreciated that the terms T cell and T lymphocyte can beinterchanged herein. The migration of the T cells is, optionally,trans-endothelial. The endothelium is, optionally, that of thepancreatic microvasculature that separates the islet cells from theblood supply.

The peptide is, optionally, an isolated peptide. The peptide may besynthesized (i.e. chemically synthesized, for instance in the same wayas a small molecule pharmaceutical) or it may be produced recombinantly,for instance in a separate cellular system (cell culture) or animal.

The amino acid sequence of the peptide that we have found to be usefulis SVTEQGAELSNEER (SEQ ID NO: 1). This sequence may be comprised withina larger peptide or protein, or a chimaeric or fusion protein.Alternatively, the peptide may consist solely of SEQ ID NO: 1. All ofthese fall within the definition of the peptide as used herein. Thepeptide according to SEQ ID NO: 1 represents amino acids 28-41 of the14.3.3 zeta/delta (14.3.3.ζδ) protein, which in turn is a 245 amino acidproduct of the YWHAZ gene.

It is also preferred that analogues or variants of the peptide can beused. Particularly preferred in this respect are analogues (or variants)based on conservative amino acid substitutions. The preferred peptide is14 amino acids long, although the peptide can also be as few as 13, 12,11 or 10 amino acids or as many as 15, 16, 17 18, 19 or 20 amino acids.Where amino acids are added or removed, these are preferably to or fromthe N and/or C terminus of the peptide. Other modifications to thechemical structure that protect the peptide from degradation orclearance in vivo are also preferred variants, for example but notrestricted to, PEGylation which utilises a linker or spacer as is knownin the art. Most preferably, any analogue should retain or improve uponthe desired function, namely the inhibition of T cell migration,compared to SVTEQGAELSNEER. This may be through changes in affinity forcognate receptor(s) or changes that alter the pharmacokinetic profile ofthe peptide in vivo. It will be appreciated that it is now within theskill of the art to modify peptide chemistry to increase thepharmacological ‘profile’ of peptides in vivo, and that these changesare not based solely on amino acid substitution.

Reference herein will be made to the peptide, but it will be understoodthat this also encompasses any analogues thereof, unless otherwiseapparent.

The action of the peptide may be as an agonist of its cognatereceptor(s).

The inhibition of the migration of the T cells may be the recruitment ofsaid cells to the pancreas, for instance from the blood. In someembodiments the inhibition of the migration of the T cells is therecruitment of said cells to secretory glands, for example salivary ortear glands. In some embodiments the inhibition of the migration of theT cells is the recruitment of said cells to salivary glands.Advantageously, this inhibition may prevent the formation of ectopictertiary lymphoid organs (TLO).

Optionally, the T cells are auto-reactive T cells. These may preferablytarget the pancreas, especially the islet cells of the pancreas. In someembodiments the auto-reactive T cells target secretory glands, forexample salivary and/or tear glands. In some embodiments theauto-reactive T cells target salivary glands. The T cells may be CD4+orCD8+.

In a particularly preferred embodiment, the peptide serves to inhibit(i.e. reduce) the recruitment of auto-reactive T cells to the islets ofthe pancreas.

In some embodiments, the peptide serves to inhibit the recruitment ofauto-reactive T cells to the salivary glands.

It will be appreciated that the peptide acts upon the individual towhich it is administered. As such, the auto-reactivity of any T cells isreactivity against self (i.e. islet cells of the pancreas) from thatindividual. The individual is a mammal, optionally, a rodent such as arat or mouse, or a primate, particularly an ape or human.

As the presence of the peptide serves to inhibit the migration of the Tcells, increasing the amount of peptide that the individual is exposedto will serve to further inhibit said migration. Optionally, the levelof inhibition of migration is such that migration is reduced by at least50% (in terms of numbers of T cells that are recruited), but mostpreferably this reduction is at least 60%, more preferably at least 70%,more preferably at least 80%, more preferably at least 90%, morepreferably at least 95%, more preferably at least 99% and mostpreferably reduced to negligible levels. Ideally, of course, no T cellswill migrate but this may not be realistic and in fact, all that isrequired is that normal function of the target tissue, for instance theislet cells, is largely preserved and/or returned (or at least as closeto normal levels as possible or desirable to alleviate the condition tobe treated).

The present peptide is most useful, therefore, in treating a number ofconditions. These include those in which T cells play a role inpathology or conditions associated with T cell auto-reactivity. Thesemay include T cell mediated chronic inflammatory disease and autoimmunedisease.

Diabetes mellitus (type 1), is particularly preferred. In someembodiments the condition is Sjogren's syndrome. Sjogren's syndrome is achronic inflammatory autoimmune disease which is characterised by T andB lymphocyte infiltration of secretory glands, for example the salivaryglands. The inventors of the present invention have found that thepresent peptide can advantageously inhibit T lymphocyte infiltration ofthe salivary glands in Sjogren's. The present peptide is thereforeenvisaged as an effective treatment for Sjogren's syndrome.

Thus, in another aspect, the present invention provides a method for thetreatment of Sjogren's syndrome by administration of a peptidecomprising N′-SVTEQGAELSNEER-C′ to a patient in need thereof.

Also envisaged are juvenile onset diabetes, rheumatoid arthritis andCrohn's disease, atherosclerosis, psoriasis, inflammatory and fibroticliver disease(s) including steatohepatitis and cirrhosis and uveitis.The peptide therefore preferably functions to treat any of the above,but most preferably type 1 diabetes (T1D). The peptide may be consideredas serving to rescue or preserve residual pancreatic function. This maybe lost function that has occurred due to attack by the auto-reactive Tcells. The peptide may be considered as serving to improve diabeticoutcomes, i.e. a reduction in the symptoms of T1D. The peptide may alsobe considered as serving to improve other morbidities associated withloss of pancreatic function which include renal (e.g. nephropathy;diabetic kidney disease), neurological (e.g. peripheral neuropathy) andcardiovascular complications (e.g. diabetic retinopathy andcardio-cerebral disease due to accelerated atherosclerosis), associatedwith the loss of pancreatic function (in turn) associated with T1D.Therefore, the peptide may be most useful in the treatment and/orprophylaxis of the above conditions, particularly T1D and itsco-morbidities (as described).

In another aspect there is provided a method for treatment of Sjogren'ssyndrome by administration, to a patient, of:

-   -   a peptide consisting of no more than 20 amino acids and        comprising N′-SVTEQGAELSNEER-C′ (SEQ ID NO: 1) or an analogue or        variant thereof that inhibits T cell migration; or    -   a chimeric or fusion protein comprising said peptide.

The invention also provides a polynucleotide sequence coding for saidpeptide, which is also useful in the treatment and/or prophylaxis of anyof the above conditions. The polynucleotide may be DNA, RNA or a DNA/RNAhybrid. This polynucleotide encodes the peptide or its analogue.Although there are a considerable number of possible combinations, weprovide at least two examples of a polynucleotide that encode the aminoacid sequence of SVTEQGAELSNEER (SEQ ID NO: 1). These are:

(SEQ ID NO: 2) 5′-AGU GUU ACU GAA CAA GGU GCU GAG UUA UCU AAU GAGGAG AGA-3′; or (SEQ ID NO: 3)5′-AGC GUC ACC GAG CAG GGC GCC GAA UUG UCC AAC GAA  GAG AGG-3′.

The above sequences are only examples, and are given in RNA form, butthe invention also provides for the DNA form (with T replacing U) andDNA/RNA hybrid form thereof, as well as the complementary sequences ofboth the RNA, DNA and RNA/DNA hybrid forms (the complementary sequencesbeing in RNA, DNA or DNA/RNA). Variants having at least 80% sequencehomology are preferred, the variant encoding a peptide that is at least50%, for instance, as efficacious as SEQ ID NO: 1. Variants have atleast functions 85% sequence homology, at least 90% sequence homology,at least 95% sequence homology, at least 99% sequence homology are alsopreferred (rounding to the nearest whole number). This may be determinedby programs such as BLAST, for instance.

Also provided is a plasmid (i.e. a construct), comprising thepolynucleotide which encodes the peptide (or its analogue or variant).The polynucleotide is preferably operably linked to a suitable promoter.The promoter may be a pancreas-specific promoter, for instance. In someembodiments the promoter is a secretory-gland-specific promoter. In someembodiments the promoter is a salivary-gland-specific promoter.

The polynucleotide encoding the peptide may be delivered byadministration of a suitable vehicle containing the polynucleotide or towhich it is bound. Examples include a so-called gene gun where thepolynucleotide may be attached to a gold particle fired through theskin. Alternatively, and more preferably, the polynucleotide (forinstance a plasmid comprising it) could be encapsulated within a viralvector or capsid. Preferred examples include adenoviral vectors. Thosethat target the pancreas or secretory glands, for example salivary ortear glands, are preferred.

Administration of the peptide may be by delivery of the peptide per se,for instance in the form of a pharmaceutically acceptable formulation,or by delivery and expression of the polynucleotide encoding thepeptide, for instance in the forms described above. These may bedelivered, for instance to the blood, by injection. This may beintramuscularly or subcutaneously. These may also be delivered via amucosa, such as the oral, nasal or rectal mucosa. These may also bedelivered in the form of a spray or tablet or in the form of asuppository. These may also be ingested orally into the stomach althoughin the case of the peptide this may require the provision of the peptidein a pro-drug form to alleviate or combat the effects of the GIdigestion.

Although useful in one aspect, it will be appreciated that that it isnot necessarily the case that that the peptide or the polynucleotideencoding it is, or needs to be, targeted at or to the pancreas (at leastfor T1D) or the salivary glands (at least for Sjogren's). Optionally,therefore, for peptide delivery, increasing systemic presentation in theblood plasma is all that is required. Delivery specifically to thepancreas is not required. Nevertheless, in an alternative embodiment,delivery specifically to the pancreas or the salivary glands may be usedas this could increase efficacy. The same applies for thepolynucleotide.

Direct targeting to the pancreas, salivary glands or tear glands isenvisaged, as part of a targeted gene therapy including thepolynucleotide encoding the peptide.

Also provided is a pharmaceutically-acceptable composition orpreparation comprising the peptide, the polynucleotide, the plasmid orthe viral vector described herein. Optionally, thepharmaceutically-acceptable composition comprises the peptide and issuitable for injection or ingestion.

As explained above, methods of treatment and/or prophylaxis of theconditions above are envisaged, particularly conditions associated withT cell mediated chronic inflammatory disease, including T cellauto-reactivity, T cell mediated chronic inflammatory disease andautoimmune disease. Diabetes mellitus (type 1), is particularlypreferred. Also envisaged are juvenile onset diabetes, rheumatoidarthritis and Crohn's disease, atherosclerosis, psoriasis, inflammatoryand fibrotic liver disease(s) including steatohepatitis and cirrhosisand uveitis, as well as any of the above-mentioned morbidities. Themethods may comprise administering to a patient in need thereof atherapeutic amount of the peptide or polynucleotide in any of themanners described herein.

Thus, provided is a method of treatment and/or prophylaxis of acondition associated with T cell mediated chronic inflammatory disease,including T cell auto-reactivity, T cell mediated chronic inflammatorydisease and autoimmune disease. In particular, the condition is diabetesmellitus (type 1). However, the condition may also be selected from thegroup consisting of: juvenile onset diabetes; rheumatoid arthritis;Crohn's disease; atherosclerosis; psoriasis; inflammatory and fibroticliver disease(s) including steatohepatitis and cirrhosis; and uveitis;or the condition may be selected from the group consisting ofnephropathy; diabetic kidney disease; peripheral neuropathy; diabeticretinopathy; and cardio-cerebral disease.

The methods may be for the treatment of said conditions or of thetreatment of said conditions. Alternatively, the methods may be for theprophylaxis of said conditions or may be of prophylaxis of saidconditions. Alternatively, the methods may be any combination thereof.

Also provided is the peptide and/or the polynucleotide encoding it foruse in the treatment and/or prophylaxis of the conditions descriedherein. Reference herein to methods includes such use.

All of the features described herein (including any accompanying claims,abstract and drawings) may be combined with any of the above aspects inany combination, unless otherwise indicated.

BRIEF DESCRIPTION OF THE FIGURES

The invention will now be described with reference to the Figures where:

FIG. 1: Adiponectin inhibits the transendothelial cell migration ofperipheral blood lymphocytes (PBL); FIG. 1A is a graph of PBLtransmigration versus adiponectin amount, FIG. 1B is a graph of PBLtransmigration versus log of adiponectin (AQ) concentration, and FIG. 1Cis another graph of PBL transmigration versus adiponectin concentration;

FIG. 2: Inhibition of AMPK with compound C restores the migration ofPBL;

FIG. 3: PBL from T1D patients are released from the inhibitory effect ofadiponectin on transendothelial cell migration; FIG. 3A is a graph ofPBL transmigration in T1D and control samples, and FIG. 3B is a graph of% inhibition of PBL transmigration in T1D and control samples;

FIG. 4: The expression of adiponectin receptors on PBL is reduced inpatients with T1D; FIG. 4A is a graph showing frequency of PBLexpressing adiponectin receptor AR1 in T1D and control samples, and FIG.4B is a graph showing frequency of PBL expressing adiponectin receptorAR2 in T1D and control samples;

FIG. 5: The expression of adiponectin receptors in T1D or healthycontrol subjects correlates with the inhibition of lymphocyte migrationby Adiponectin; FIG. 5A is a graph of % inhibition of transmigrationversus frequency of AR1 expression, and FIG. 5B is a graph of %inhibition of transmigration versus frequency of AR2 expression;

FIG. 6: The expression of adiponectin receptors on different leukocytesubsets; FIG. 6A is a graph showing frequency of AR1 expression fordifferent cell types, and FIG. 6B is a graph showing frequency of AR2expression for different cell types;

FIG. 7: B cells mediate the adiponectin-induced inhibition of T cellmigration; FIG. 7A is a graph of transmigration for various cellsamples; FIG. 7B is another graph of transmigration for various cellsamples;

FIG. 8: B cells modulate PBL transmigration through secretion of apeptide;

FIG. 9 shows the sequence of the secreted peptide and different isoformsof the 14.3.3 proteins;

FIG. 10: Comparison of MS/MS parent ion m/z 774.88 from B cellsupernatants and a synthetic version of the peptide;

FIG. 11: The peptide inhibits T cell migration across endothelial cellsin vitro; FIG. 11A is a graph of PBL transmigration for various samples,and FIG. 11B is a graph showing PBL transmigration versus logconcentration;

FIG. 12: Absolute number of T cells in the inflamed peritoneum of wildtype or B cell knockout mice in the presence or absence of the peptide.

FIG. 13: The effect of adiponectin (Aq) on the transendothelial cellmigration of peripheral blood lymphocytes.

(FIG. 13A) Dose response with an EC50 of ≈40 nM conducted in staticadhesion assay.

(FIG. 13B) The effects of 15 μg/ml Aq on lymphocyte migration in a flowbased assay (mimics the flow of blood).

(FIG. 13C) The inhibitory pathway is effective on endothelial cellsisolated form different tissues (HUVEC=Umbilical cord; HSAVEC=saphenousvein; HSEC=liver sinusoidal endothelial cells; HDMEC=Dermalmicrovascular endothelium).

(FIG. 13D) The effects of an AMP-kinase inhibitor on the effects ofadiponectin. AMPK is a signalling adapter that is required foradiponectin-receptor signalling.

FIG. 14: B cells are required for the adiponectin mediated inhibition ofT cell trafficking. 15 μg/ml of Aq significantly reduced lymphocytemigration across endothelial cells. removing B cells from the peripheralblood lymphocyte preparation completely inhibited this response. Thiscould be reconstituted using supernatants from Aq stimulated B cellscould also effectively inhibit lymphocyte migration, but this effect waslost when supernatants were prepared in the presence of Brefeldin-A, aninhibitor of B cell secretion. These data demonstrate that a solublemediator released from B cells is required.

FIG. 15: a 14 amino acid peptide released from B cells regulates T celltrafficking.

FIG. 16: PEPITEM inhibits T cells transmigration.

16A) The synthetic peptide was highly effective at inhibiting thetransmigration of lymphocytes, while control peptides, including ascrambled version (randomized reorganisation of the native peptidesequence), were ineffective at inhibiting lymphocyte migration.

16B) The peptide had an EC50 of ≈20 pM. As it effectively inhibitedlymphocyte migration across endothelial cells, we called the agentPEPtide Inhibitor of Trans Endothelial Migration; “PEPITEM.”

FIG. 17: PEPITEM inhibits T cell migration AND promotes the recruitmentof anti-inflammatory regulatory T cells. PEPITEM inhibits T cellmigration across EC with the same pattern as adiponectin (FIG. 17A); Itis effective at inhibiting the migration of memory CD4+ and CD8+ Tcells, but it has no effect neutrophils, or monocytes (including CD16−and CD16+ subsets. Naïve lymphocytes were not assessed in this analysisas they do not adhere to the endothelial cell monolayer (FIG. 17B).Interestingly, the efficiency of the migration of regulatory T cells(T-regs), which have anti-inflammatory functions, was increased byPEPITEM (FIG. 17C).

FIG. 18: PEPITEM does not directly regulate T cell migration. Again themost obvious mode of action of PEPITEM was by directly regulating themigratory functions of T cells. However, this was not the case. When PBLwere treated with PEPITEM and the agent was washed away prior to assayon endothelium, the efficiency of lymphocyte migration was not effected.However, pre-treating the endothelial cells with PEPITEM resulted ininhibition of lymphocyte trafficking. Thus, PEPITEM operates bystimulating endothelial cells to release an agent that inhibits T celltrafficking.

FIG. 19: The induction of sphingosine-1-phosphate (SIP) synthesis byendothelial cells inhibits T cell migration. As PEPITEM did not directlyinhibit lymphocyte migration we tested the hypothesis that a knownregulator of lymphocyte trafficking in other tissues,sphingosine-1-phosphate (S1P), was the terminal step in this pathway.

(FIG. 19A) A S1P-receptor antagonist (W146), releases lymphocytes fromthe inhibitory effects of Adiponectin.

(FIG. 19B) A S1P-receptor antagonist (W146), releases lymphocytes fromthe inhibitory effects of PEPITEM.

(FIG. 19C) Addition of exogenous S1P dose dependently inhibits T cellmigration.

(FIG. 19D) Endothelial cell express sphingosine kinase-1 (SPHK1) but notsphingosine kinase-2 (SPHK2).

(FIG. 19E) An inhibitor of SPHK1 releases lymphocytes from theinhibitory effects of PEPITEM.

FIG. 20: S1P regulates the affinity of the lymphocyte integrin LFA-1(CD11a/CD18; αLβ2) when the cells are immobilised on ICAM and activatedwith IP10 (CXCL10)

(FIG. 20A) KIM127 for the intermediate affinity site and

(FIG. 20B) antibody 24 for the high affinity epitope on memory T cellstreated with S1P. FIG. 21: Absolute number of T cells in the inflamedperitoneum of wild type or B cell knockout mice in the presence orabsence of the peptide.

FIG. 21A The recruitment of T cells into the peritoneum of Jh−/− (B-cellknockout animals) was greater at baseline (i.e. after challenge withPBS) than wild type animals. After challenge with intraperitonealinjection with zymosan T cells Numbers increased in the wild typeanimals. There was a dramatic and significant increase I T cell numberin the B cell knockout mice and this was significantly reduced in thepresence of PEPITEM, but not the scrambled peptide.

FIG. 21B The recruitment of T cells into the liver of B-cell knockoutanimals after challenge with none-typhoidal salmonella infectionincreased when compared to the wild type animals.

FIG. 22: The adiponectin/PEPITEM pathway is altered in patients withtype 1 diabetes. In type-1-diabetic patients the expression ofadiponectin receptors is significantly reduced compared to healthy agedmatched controls (FIGS. 22A and 22B). The inhibition of lymphocytetrafficking by adiponectin correlates significantly with the level ofexpression of adiponectin receptors on B cells (FIG. 22C), so thatpatient and healthy cohorts separate Into discrete clusters on thecorrelation graph. Importantly, although patient lymphocyte arerefractory to stimulation by adiponectin (FIG. 22D), the inhibitorypathway can be recapitulated for these cells by addition of exogenousPEPITEM.

FIG. 23: PEPITEM significantly reduces the number of infiltrating Tcells into the salivary glands of a mouse model of Sjogren's syndrome;FIG. 23A is a graph showing the number of T cells in the salivary glandsof different mice, and FIG. 23B shows immunofluorescentiv stained slidesof the salivary glands of different mice.

FIG. 24: PEPITEM reduces the mRNA for inflammatory cytokines in thesalivary glands of a mouse model of Sjogren's syndrome.

DETAILED DESCRIPTION

WO2007127935 relates to the histone deacetylase, HDAC7. It sets out toidentify the phosphatase that dephosphorylates HDAC7 and finds that anumber of proteins bound to HDAC7, including the peptide describedherein as SEQ ID NO NO: 1. The focus of the document is that a “targetsubunit” of the myosin phosphatase (MYPT1) also bound HDAC7 and as suchthe teaching is directed to the interaction between HDAC7 and MyosinPhosphatase via this subunit of myosin phosphatase. There is no mentionthat our peptide has any value, nor that it interferes with theHDAC7—Myosin Phosphatase interaction. US2002164668 (A1) andUS20030064411 (A1) disclose our peptide and pharmaceuticalpreparations/compositions comprising it in relation to the treatment ofAlzheimer's disease. US20040053309 (A1) also discloses our peptide, butrelates to the identification of proteins and protein isoforms that areassociated with kidney response to toxic effectors. However, none of theprior art discloses the use of our peptide or analogues thereof.

We have been interested in the ability of the adipocyte derivedcytokine, adiponectin, to regulate the recruitment of human T cells toinflamed endothelium. Previously, adiponectin deficient mice were shownto have a two-fold increase in leukocyte adhesion to endothelial cellsand importantly, leukocyte recruitment was normalized by the addition ofrecombinant adiponectin. In our in vitro studies we used statictranswell assays, as well as flow based adhesion assays, to track themigration of T cells (which were in crude isolates of peripheral bloodlymphocytes [PBL]) across TNF-α and IFN-γ stimulated endothelial cells.T cell migration was dose dependently blocked by adiponectin (FIG. 1).

The effect of adiponectin on T cell transmigration was mediated bysignalling through the adiponectin receptors (AR1 and AR2).AMP-activated protein kinase (AMPK) is a crucial intermediate in thedown stream signalling from AR1 and AR2 and when PBL were pre-treatedfor 30 minutes with the AMPK inhibitor, compound C, the effects ofadiponectin on the inhibition of T cell migration were ablated, i.e. Tcell migration returned to the levels observed in the absence ofadiponectin (FIG. 2). Compound C did not have any effects on migrationin the absence of adiponectin.

Importantly, we found that the adiponectin mediated inhibition of T cellmigration was significantly compromised in patients with T1D i.e., theability of adiponectin to modulate T cell recruitment in our in vitromigration assays was lost when PBL isolated from T1D were used (FIG. 3).We have now shown that both AR1 and AR2 are significantly down regulatedon lymphocytes in T1D (FIG. 4), and the levels of adiponectin mediatedinhibition of T cell migration in vitro correlate exquisitely withexpression of these receptors in T1D, to the extent that patient andhealthy control cohorts cluster independently when receptor density isplotted against sensitivity to adiponectin in the endothelial celltransmigration assay (FIG. 5).

We do not believe that adiponectin represents a suitable target forregulating T cell recruitment in T1D. Its concentration in thecirculation is not altered in T1D, indicating that aspects ofadiponectin biology other than its bioavailability are importantarbiters of function. Moreover, adiponectin is a pleiotropic agent withimportant roles in metabolic homeostasis, raising the possibility ofserious off target side effects.

Rather, we believe that targeting pathways down stream of adiponectin,which regulate T cell migration, would provide a therapeutic modality ofgreater precision. Thus, we have now gone on to show unequivocally thatadiponectin achieves its effects on T cell migration by the induction ofa novel mediator, which we believe is a peptide inhibitor oftrans-endothelial migration that is released from B lymphocytes.Importantly, B lymphocytes express adiponectin receptors, so can respondin an appropriate manner to stimulation by this agent (FIG. 6).

Moreover, the inhibition of T cell migration by adiponectin is lost if Bcells are removed from mixed lymphocyte preparations (PBL), andinhibition of T cell migration is regained if isolated B cells are addedto purified preparations of T cells (FIG. 7a ). Interestingly, naturalkiller lymphocytes (NK cells), which also express high levels ofadiponectin receptors (FIG. 6) are not capable of regulating themigration of T cells (FIG. 7b ), indicating that the regulation of Tcell migration is mediated exclusively by B lymphocytes and not othercellular components of the PBL population.

B cells mediate their effects in this system by secretion of thepeptide. Thus, supernatants conditioned by adiponectin stimulated Bcells, could effectively inhibit T cell migration (FIG. 8). Moreover,the effects of conditioned supernatants were lost when Brefeldine A,which is an inhibitor of B cell secretory pathways, was used to inhibitthe release of the peptide from B cells in to the conditioned medium,see (FIG. 8).

We have now definitively identified the secreted peptide released from Bcells in response to adiponectin stimulation. Using mass spectrometricanalysis adiponectin conditioned B cell supernatant, as well as therelevant control supernatants were purified and analysed by LC-MS/MS.Comparative analysis of a protein sequence database revealed a singlecandidate peptide unique to the adiponectin conditioned B cellsupernatant, described in Table 1, below.

TABLE 1 Candidate peptide for the peptide revealed by comparativeanalysis of B cell supernatants Elution time Association m/z (min) ScoreModification protein Sequence 774.88 13.2 63.1 NA 14-3-3  SVTEQGAELSNEERzeta/delta

Due to the statistically stringent nature of the fragmentation analysis,the software was able to provide a definitive sequence with a highprobability of accuracy and to identify the 14.3.3 zeta/delta(14.3.3.ζδ) protein as the precursor protein. Indeed the peptiderepresents amino acids 28-41 of the 14.3.3 ζδ protein, which in turn isa 245 amino acid product of the YWHAZ gen. Stringent database searchesdemonstrate that the peptide sequence is unique to this protein and isnot shared, even by the other six members of the 14.3.3 family ofproteins (FIG. 9). The peptide is not a member of any known family ofimmuno-regulatory molecules and due to its chemistry, has attractivetherapeutic potential.

We have been able to successfully synthesise the peptide. Comparativeanalysis of the B cell derived peptide and the synthetic version showidentical mass: charge ratios in mass spectrometry analysis, showingthat the native peptide has not been subject to post-translationalmodification prior to excision from the 14.3.3. zeta/delta protein andsecretion from B cells (FIG. 10).

The peptide has efficacy both in vitro and in vivo. Using the syntheticpeptide we constructed a dose response curve in our in vitro assay of Tcell migration (FIG. 11). The peptide has an EC50 of ≈20 pM in thisassay. We have also utilised the peptide in an in vivo model of acute,zymosan induced peritonitis (FIG. 12). In this model we first showedthat the knockout of B lymphocytes (the cellular source of the peptide)resulted in an increase in the recruitment of T lymphocytes into theperitoneal cavity. We then conducted the experiment after injection ofthe peptide into the blood and peritoneum cavity of the B cell knockoutmice. The peptide was able to significantly reduce the recruitment of Tcells to the peritoneum after challenge with zymosan (FIG. 12).

Without being bound by theory, we understand that the followingrepresents the paradigm by which PEPITEM regulates T cell traffickingacross endothelial cells during inflammation: Adiponectin, operatingthrough the receptors Adipo-R1 and Adipo-R2 (AR1/2), stimulates therelease the immune-regulatory peptide, PEPITEM, from B cells, which arerecruited to the endothelial cell surface during inflammation. PEPITEMstimulates endothelial cells through its cognate receptor, promoting theformation and release of sphingosine-1-phosphate (S1P). S1P in turnstimulates T cells recruited to the endothelial cell surface duringInflammation through the S1P-receptor(s) S1PR1/4, a signal that inhibitsthe ability of T cells to traffic across the endothelial cell barrierand enter inflamed tissue.

The following Examples present experimental proofs for the function ofthis pathway in both in vitro and in vivo studies, demonstrate changesin pathway function associated with chronic auto-immune disease inhumans, and describe the identity the PEPITEM peptide.

EXAMPLE 1 Adiponectin Inhibits the Transendothelial Cell Migration ofPeripheral Blood Lymphocytes (PBL).

Endothelial cells were cultured in low serum medium and stimulated withTNF-α/IFN-γ for 24 hours in the absence of adiponectin. PBL wereisolated and treated with adiponectin at 0.0001 to 15 μg/ml for onehour.

The results are shown in FIG. 1, where part (A) shows that PBLtransmigration was significantly and dose dependently reduced byadiponectin in a static adhesion assay; part (B) shows that adiponectinhad an EC50 of 0.94 μg/ml as determined by linear regression; and part(C) shows that Adiponectin was equally effective at inhibiting PBLmigration in a flow based adhesion assay. Data is representative of atleast three independent experiments and were analysed using t-test,one-way ANOVA and Dunnett's multiple comparisons post-test. *p≦1.01,**p≦1.001, ***p≦1.0001.

Inhibition of AMPK with Compound C Restores the Migration of PBL.

Endothelial cells were cultured in low serum medium and stimulated withTNF-α/IFN-γ for 24 hours. Compound C was added to PBL at 10 μg/ml for 30minutes prior to addition of adiponectin at 15 μg/ml for 1 hour.Adiponectin treatment induced a decrease of transmigration, which wasrestored to normal, control levels in the presence of compound C. Theresults are shown in FIG. 2, where data is representative of threeexperiments and were analysed using one-way ANOVA and Dunnet's multiplecomparisons post-test. **p≦0.001, ***p≦0.0001.

PBL from T1D Patients are Released from the Inhibitory Effect ofDdiponectin on Transendothelial Cell Migration.

Endothelial cells were cultured in low serum medium and stimulated withTNF-α/IFN-γ for 24 hours in absence of adiponectin. The results areshown in FIG. 3. Part A) shows that Adiponectin-mediated inhibition ofPBL transmigration is lost in T1D; and part B) shows that the percentageof inhibition was calculated by dividing the percentage oftransmigration with adiponectin treatment by the percentage oftransmigration of untreated PBL. n=13 for HC groups and n=12 for T1Dgroup. Data was analysed using t-test and one-way ANOVA and Bonferonni'smultiple comparisons post-test. ***p≦1.0001.

The Expression of Adiponectin Receptors on PBL is Reduced in Patientswith T1D.

The frequency of PBL expressing adiponectin receptors AR1 or AR2 weredetermined for each healthy or diseased subject and are shown in FIGS.4A) and 4B), respectively. Data is represented as mean±SEM and wasanalysed using t-test or Mann Whitney t-test when data did not pass theKolmogorov-Smirnov normality test.

The Expression of Adiponectin Receptors in T1D or Healthy ControlSubjects Correlates with the Inhibition of Lymphocyte Migration byAdiponectin.

FIG. 5A) shows the correlation between the expression of AR1 andinhibition of lymphocyte migration, whilst FIG. 5B) shows thecorrelation between the expression of AR2 and inhibition of lymphocytemigration. Correlations were determined using linear regressionanalysis.

The Expression of Adiponectin Receptors on Different Leukocyte Subsets.

FIGS. 6A) and B) show the expression of AR1 (FIG. 6A) and AR2 (FIG. 6B)on different cell types. Data is mean ±SEM and are representative ofseven healthy controls. Data was analysed using one-way ANOVA andBonferonni's multiple comparisons post-hoc test. ***p≦0.0001.

B Cells Mediate the Adiponectin-Induced Inhibition of T Cell Migration.

FIG. 7A) shows that the migration of PBL is lost when they are depletedof B cells (Bs) and regained when B cells are added back to isolated Tcells. FIG. 7B) shows that the migration of natural killer cells is notaffected by adiponectin and addition of NKs to T cells does not regulatethe migration of the T cells. Data is mean ±SEM and are representativeof at least three independent experiments. Data was analysed usingone-way ANOVA and Bonferroni's multiple comparisons post-test.**p≦1.001, ***p≦1.0001.

B Cells Modulate PBL Transmigration Through Secretion of a Peptide.

B cells were isolated and incubated in presence or absence ofadiponectin at 15 μg/ml. Supernatant was taken after one hour and addedto Bs-ve PBL which significantly restored the adiponectin inhibition ofPBL transmigration. For some experiments, B cells were treated withBrefeldin A, an inhibitor of B cell secretion. These supernatants werenot able to regulate the migration of T cells. This is shown in FIG. 8,where the data is shown as mean ±SEM and is representative of threeindependent experiments analysed using one-way ANOVA and Bonferroni'smultiple comparison post test. ***p<0.001, ns=non-significant.

The sequence of the peptide was determined and it is shown in FIG. 9together with the different isoforms of the 14.3.3 proteins. See alsoTable 1 above.

Comparison of MS/MS Parent Ion m/z 774.88 from B Cell Supernatants and aSynthetic Version of the Peptide.

The ion m/z 774.88 is a fragmentation product of the analysis protocoland is generally only of use for identification using MS/MS, but can bean important parameter for comparison.

A comparison of the Mass Spec profiles of parent ion m/z 774.88 from Bcell supernatants and a synthetic version of the peptide analysis isshown in FIG. 10, revealing identical mass: charge ratios. Thisconfirmed sequence identity and showed that the peptide is not subjectto post-translational modification prior to secretion.

The Peptide Inhibits T Cell Migration Across Endothelial Cells In Vitro.

PBL were treated with Adiponectin (15 μg/ml as positive control) or thepeptide at concentrations between 0.001 and 10 ng/ml, a scramble peptidewas used as a negative control (used 10 ng/ml). Other bioactive peptideswere also used to demonstrate specificity of the peptide (i.e. tetanustoxoid peptide (TTp) at 10 ng/ml and pro-insulin (P1) at 10 ng/ml). Theresults are shown in FIG. 11. FIG. 11A) shows that PBL transmigrationwas dose-dependently reduced in presence of the peptide but not in thepresence of the scrambled peptide, TTp or PI controls. FIG. 11B) showsthat the EC50 of the peptide (18.6 pM) was calculated using non linearregression analysis. Data is representative of three independentexperiments and was analysed using one-way ANOVA and Bonferroni'smultiple comparison post test. *p≦1.01, **p≦1.001, ***p≦1.0001.

Absolute Number of T Cells in the Inflamed Peritoneum of Wild Type or BCell Knockout Mice in the Presence or Absence of the Peptide.

Leukocytes were collected from the peritoneum after 48 hours injectionof zymosan (or PBS as control) with or without the peptide or ascrambled peptide. T cells were identified by expression of CD3. Thepeptide or a scrambled peptide was injected at a final concentration of300 μg/mouse. The results are shown in FIG. 12, where data for eachgroup is the mean and was analysed using one-way ANOVA and Bonferroni'smultiple comparisons post-test. *p≦0.01.

EXAMPLE 2

This Example shows the results of further work undertaken and thuscompliments Example 1.

The Effect of Adiponectin (AQ) on the Transendothelial Cell Migration ofPeripheral Blood Lymphocytes (PBL).

Refer to FIG. 13. Endothelial cells were cultured in low serum mediumand stimulated with TNF-α/IFN-γ for 24 hours in the absence ofadiponectin. PBL were isolated and treated with adiponectin at 0.0001 to15 μg/ml for one hour. Part (A) shows that PBL transmigration wassignificantly and dose dependently reduced by adiponectin in a staticadhesion assay and that adiponectin had an EC50 of ˜40 nM as determinedby linear regression; and part (B) shows that Adiponectin was equallyeffective at inhibiting PBL migration in a flow based adhesion assay;and part (C) shows that Adiponectin is effective on endothelial cellsisolated from different tissues such as HUVEC (Umbilical cord), HSEC(liver sinusoidal endothelial cells) and DMEC (Dermal microvascularendothelium) but not HSAVEC (saphenous vein). In part (D), compound C,an AMP-kinase inhibitor, was added to PBL at 10 μg/ml for 30 minutesprior to addition of adiponectin at 15 μg/ml for 1 hour. AMPK is asignalling adapter that is required for adiponectin-receptor signalling.Adiponectin treatment induced a decrease of transmigration, which wasrestored to normal control levels in the presence of compound C. Thesedata indicate that adiponectin has a strong capacity to regulate thetransmigration of lymphocytes through action on its receptors expressedon PBL. Data is a pool of at least three independent experiments andwere analysed using t-test, one-way ANOVA and Dunnett's multiplecomparisons post-test. **p≦1.01, ***p≦1.001.

T Cells do not Posses Adiponectin Receptors.

The simplest interpretation of the previous experiment is that T cellsare under the direct control of Aq. However, T cells lack theappropriate receptors. However, other leukocytes do have Adipo-R1/2 andboth monocytes and B cells show high levels of expression.

Expression of both adiponectin receptors, AdipoR1 and AdipoR2, wasmeasured on PBMC by flow cytometry using rabbit anti-human adiponectinreceptor 1 and 2 antibodies (Phoenix peptides). Adiponectin receptorexpression is shown on the horizontal axis against pan markers of PBMCsub-populations (vertical axis). AdipoR1 and AdipoR2 are highlyexpressed on monocytes (CD14+) and on B cells (CD19+) but at very lowlevels on T cells (CD3+). This indicates that adiponectin cannotdirectly control T cell migration.

B Cells are Required for the Adiponectin Mediated Inhibition of T CellTrafficking.

Refer to FIG. 14. Endothelial cells were cultured in low serum mediumand stimulated with TNF-α/IFN-γ for 24 hours in the absence ofadiponectin. PBL transmigration was measured after removal of B cellsusing bead positive selection and after reconstitution with B cells thatwere isolated using bead negative selection in presence or absence ofadiponectin (15 μg/ml). Supernatants from adiponectin-treated B cells orB cell treated with Brefeldin A to block protein secretion were added toPBL.

Removing B cells form the peripheral blood lymphocyte preparationcompletely inhibited this response. This could be reconstituted usingsupernatants from Adiponectin stimulated B cells that could alsoeffectively inhibit lymphocyte migration, but this effect was lost whensupernatants were prepared in the presence of Brefeldin-A, an inhibitorof B cell secretion. These data demonstrate that a soluble mediatorreleased form B cells is required.

Data is a pool at least three independent experiments and was analysedusing one-way ANOVA and Bonferroni's multiple comparison post test.*p≦0.05, ***p≦0.001.

A 14 Amino Acid Peptide Released from B Cells Regulates T CellTrafficking

Refer to FIG. 15. B cells were isolated using negative selection andincubated with adiponectin for an hour. Supernatants were recovered andpurified on a C18 columns to remove large size proteins and acquired bymass spectrometry. The proteomic analysis using mass spectrometry ofsupernatants from AQ stimulated B cells revealed a 14 amino acid peptidewith the sequence SVTEQGAELSNEER. Comparing this to an in silico libraryof published and predicted sequences, the peptide demonstrated exactsequence homology to a single human protein, and represents amino acids28-41 of the 14.3.3 zeta/delta (14.3.3.ζδ) protein, which in turn is a245 amino acid product of the YWHAZ gene. The peptide is not a member,nor is it related to, nor does it have sequence similarity to, any ofthe known families of immune-regulatory peptides. Analysis of syntheticpeptide by mass spectrometry showed an identical mass:charge ratio tothe native peptide (m/z=774.88), demonstrating that the B-cell derivedproduct was not subject to any form of post translational modificationprior to release. These data indicate that the 14 amino acid peptideidentified is the mediator released by B cells under adiponectinstimulation.

PEPITEM Inhibits T Cells Transmigration

Refer to FIG. 16. Endothelial cells were cultured in low serum mediumand stimulated with TNF-α/IFN-γ for 24 hours in the absence ofadiponectin. PBL were treated with Adiponectin (15 μg/ml as positivecontrol) or the peptide at concentrations between 0.001 and 10 ng/ml, ascramble peptide was used as a negative control (10 ng/ml). Otherbioactive peptides were also used to demonstrate specificity of thepeptide (i.e. tetanus toxoid peptide (TTp) at 10 ng/ml and pro-insulin(P1) at 10 ng/ml). Part (A) shows that PBL transmigration wasdose-dependently reduced in presence of the peptide but not in thepresence of the scrambled peptide, TTp or PI controls. Part (B) showsthat the EC50 of the peptide (18.6 pM) was calculated using non linearregression analysis. The data indicates that PEPITEM is able to inhibitPBL transmigration similarly to adiponectin. Data is a pool of at leastthree independent experiments and was analysed using one-way ANOVA andBonferroni's multiple comparison post test. *p≦1.05, **p≦1.01,***p≦1.001.

PEPITEM Inhibits T Cell Migration and Promotes the Recruitment ofAnti-Inflammatory Regulatory T Cells

Refer to FIG. 17. Endothelial cells were cultured in low serum mediumand stimulated with TNF-α/IFN-γ for 24 hours in the absence ofadiponectin. PBL and the different subsets and PEPITEM were added todifferent endothelial cells and transmigration was measured. Thedifferent subsets were isolated using negative selection for Treg, CD4+and CD8+ memory and naïve T cells. Positive selection was used toisolate the different monocyte subsets.

Part (A) shows that PEPITEM inhibits T cell migration across EC with thesame pattern as adiponectin on different endothelial cell type. Part (B)shows that PEPITEM is effective at inhibiting the transmigration ofmemory CD4+ and CD8+ T cells, but it has no effect neutrophils, ormonocytes (including CD16− and CD16+ subsets. Naïve lymphocytes were notassessed in this analysis as they do not adhere to the endothelial cellmonolayer. Part (C) shows the efficiency of the migration of regulatoryT cells (Treg), which have anti-inflammatory functions, was increased byPEPITEM. These data indicate that PEPITEM is able to specificallymodulate transmigration of memory T cells and Treg.

Data is a pool of at least three independent experiments and wasanalysed using t-test and one-way ANOVA and Bonferroni's multiplecomparison post test. *p≦0.05, **p≦0.01, ***p≦0.001.

PEPITEM does not Directly Regulate T Cell Migration

Refer to FIG. 18. Endothelial cells were cultured in low serum mediumand stimulated with TNF-α/IFN-γ for 24 hours in the absence ofadiponectin. PEPITEM was added with the PBL on the endothelial cells orendothelial cells were pre-treated with PEPITEM and PBL added afterwashes or PBL were pre-treated with PEPITEM, washed and added to theendothelial cells.

When PBL were treated with PEPITEM and the agent was washed away priorto assay on endothelium, the efficiency of lymphocyte migration was notaffected. However, pre-treating the endothelial cells with PEPITEMresulted in inhibition of lymphocyte trafficking. These data indicatethat PEPITEM operates by stimulating endothelial cells to release anagent that inhibits T cell trafficking.

Data is a pool of three independent experiments and was analysed usingpaired t-test *p≦1.05, **p≦1.01.

The Induction of Sphingosine-1-Phosphate (S1P) Synthesis by EndothelialCells Inhibits T Cell Migration.

Refer to FIG. 19. PBL or B cell depleted PBL transmigration acrossIFN-γ/TNF-α treated HUVEC was measured after blockade of S1P signallingusing S1PR antagonist (W146, 10 μM) in presence or absence of (part A)adiponectin (15 μg/ml) or (part B) PEPITEM. B cell depleted PBL werepre-treated with S1P at different concentrations (0-100 μM) andtransmigration across IFN-γ/TNF-α treated HUVEC was measured (part C).Levels of SPHK1 and SPHK2 mRNA expression determined by real-time PCR ofRNA from B cells and HUVEC (part D, n=2). PBL transmigration wasmeasured across IFN-γ/TNF-α treated HUVEC pre-treated with SPHK1specific inhibitor (5 μM) in presence of PEPITEM (10 ng/ml) (part E).

The data shows that antagonism of the S1P receptor on T cells results inloss of adiponectin and PEPITEM inhibition on T cell transmigration(part A, B). Part (C) shows that addition of S1P to B cell depleted Tcells restores the inhibition of transmigration; and part (D) shows highexpression of S1P kinase 1 and 2 in HUVEC (SPHK1 and 2); and part (E)shows that inhibition of SPHK1 releases lymphocytes from the inhibitoryeffect of PEPITEM. These data indicates that PEPITEM stimulatesendothelial cells to release S1P, which in turn inhibits lymphocytetransmigration.

Data is a pool of at least three independent experiments and wasanalysed using t-test and one-way ANOVA and Bonferroni's multiplecomparison post test. *p≦0.05, **p≦0.01, ***p≦0.001.

S1P Regulates the Affinity of the Lymphocyte Integrin LFA-1.

Refer to FIG. 20. 96 well plates were coated with 50 μg/ml ofrecombinant ICAM overnight at 4° C. The plate was blocked using PBS 4%BSA for an hour at room temperature and PBL treated with IP-10 (10ng/ml) and/or S1P (10 μM) were added for 30 minutes. Excess of unbound

PBL was washed and PBL were labelled for the intermediate affinity siteof the lymphocyte integrin LFA-1 (CD11a/CD18; αLβ2) using the KIM127antibody (10 ug/ml) and for the high affinity site using antibody 24 (10ug/ml) at 4° C. The expression of both affinity site was measured onmemory T cells using mean fluorescence intensity (MFI). The data showsthat the expression of both intermediate and high affinity sitesincreased upon IP-10 stimulation is down-regulated in presence of S1P.The data indicates that S1P regulate lymphocyte transmigration bymodulating the affinity of the integrin LFA-1 that is essential tolymphocyte transmigration. Data is a pool of two independentexperiments.

Absolute Number of T Cells in the Inflamed Peritoneum of Wild Type or BCell Knockout Mice in the Presence or Absence of the Peptide.

Refer to FIG. 21. In part (A), wild-type or B cell knock-out (Jh−/−)BALB/c mice were injected with 100 ug zymosan. Leukocytes were collectedfrom the peritoneum after 48 hours injection of zymosan (or PBS ascontrol) with or without the peptide or a scrambled peptide. T cellswere identified by expression of CD3. The peptide or a scrambled peptidewas injected at a final concentration of 300 μg/mouse. The results areshown in part (A), where data for each group is the mean and wasanalysed using one-way ANOVA and Bonferroni's multiple comparisonspost-test. *p≦1.01. In part (B), wild-type or B cell knock-out C56BL/6mice were injected with Salmonella typhirium. After 5 days, liver werecollected and sections stained for T cells. The data in part (b) showsthe number of T cells per infection loci in liver sections.

The data shows that absence of B cells in mouse results in higherrecruitment of T cells in the peritoneum upon zymosan-inducedinflammation and Salmonella infection. This is reduced in the zymosantreated B cell knock-out mice by PEPITEM but not by the scrambledcontrol. These data indicates that B cells are essential to regulaterecruitment of T cells during inflammation in vivo by release of PEPITEMat sites of inflammation.

The Adiponectin/PEPITEM Pathway is Altered in Patients with Type 1Diabetes

Refer to FIG. 22. The frequency of PBL expressing adiponectin receptorsAR1 or AR2 were determined for each healthy or diseased subject by flowcytometry and are shown in part (A) and (B), respectively. Data isrepresented as mean ±SEM and was analysed using t-test or Mann Whitneyt-test when data did not pass the Kolmogorov-Smirnov normality test.Endothelial cells were cultured in low serum medium and stimulated withTNF-α/IFN-γ for 24 hours in the absence of adiponectin. PBL wereisolated from healthy controls and patients with type 1 diabetes andtreated with adiponectin 15 μg/ml for one hour. Part (C) shows acorrelation between the expression of AdipoR2 and inhibition oflymphocyte migration, Correlations were determined using linearregression analysis. Part (D) shows the transmigration of PBL from newlydiagnosed patient with type 1 diabetes, pre-treated with adiponectin orPEPITEM (n=5). Data was analysed using t-test **p<0.01.

The results show in part (A and B), lower expression of both adiponectinreceptors (AdipoR1/2) on PBL from patients with type 1 diabetes; andpart (B) shows that the lower expression of AdipoR2, the lower is thecapacity of adiponectin to inhibit lymphocyte transmigration; and inpart (D), PEPITEM was still able to inhibit lymphocyte transmigration.

The data indicates that lymphocytes from patients with type 1 diabetesare released from the inhibitory effects of adiponectin because theyexpress lower adiponectin receptors and this can be restored byexogenous addition of PEPITEM.

EXAMPLE 3

This Example shows the results of further work undertaken directed toSjogren's syndrome.

PEPITEM Significantly Reduces the Number of Infiltrating T Cells intothe Salivary Glands of Sjogren's Syndrome

Primary Sjogren's syndrome (pSS) is a chronic inflammatory autoimmunedisease with a prevalence of 0.5% in the general population. It ischaracterised by loss of function of salivary glands and autoantibodyproduction. One third of the patients present signs of extra-glandularinvolvement that extend from cutaneous vasculitis to peripheralneuropathy or pulmonary involvement. Systemic manifestations are mostcommonly observed in immunologically active patients characterised byhigh titres of autoantibody production. Lymphocyte infiltration of thesalivary glands and formation of organised inflammatory aggregates of Tand B-lymphocytes represent the histological hallmark of the disease.Acquisition of Sjogren's Syndrome is associated with the formation ofectopic tertiary lymphoid organs (TLO) which are associated with asignificant increase in the risk of developing lymphoma.

C57BL/6 mice were intraductally cannulated with 10⁸-10⁹ pfu ofluciferase-encoding replication-defective adenovirus (ADVS) to virallyinduce TLO. A group of 5 mice were administered (i.p injections) 100 μgPEPITEM and another group of 5 mice were administered 100 μg scrambledpeptide every day until day 5 post-cannulation. Cannulated salivaryglands were harvested and chopped into small pieces and digested for 20minutes at 37° C. Cells were filtered and washed, then stained for flowcytometry (FIG. 23A).

Salivary glands from mice were harvested, snap frozen, left to dryovernight at room temperature and then stored at −80° C. until use.Slides were immunofluorescently stained after being brought to roomtemperature (Figure B).

The data shows that PEPITEM administration significantly reduces thenumber of infiltrating T cells into the salivary glands of a mouse modelof Sjogren's and their organisation into ectopic lymphoid structures(TLO). These data indicate that PEPITEM administration can be used toreduce the infiltration of T cells at sites of inflammation in Sjogren'ssyndrome.

PEPITEM Reduces the mRNA for Inflammatory Cytokines in the SalivaryGlands of a Mouse Model of Sjogren's Syndrome

Salivary glands from the mice were removed and mRNA isolated usingstandard protocols. qPCR analysis of the isolated mRNA revealed thatPEPTIEM reduces the expression of cytokines which are inflammatorydrivers of disease (FIG. 24).

What is claimed is:
 1. A method for treatment of Sjogren's syndrome byadministration, to a patient, of: a peptide consisting of no more than20 amino acids and comprising N′-SVTEQGAELSNEER-C′ (SEQ ID NO: 1) or ananalogue or variant thereof that inhibits T cell migration; or achimeric or fusion protein comprising said peptide.
 2. A methodaccording to claim 1, wherein the T cells are auto-reactive T cells. 3.A method according to claim 2, wherein the peptide serves to inhibit therecruitment of auto-reactive T cells to the salivary glands.
 4. A methodaccording to claim 1, wherein a polynucleotide sequence encoding thepeptide or analogue thereof is administered.
 5. A method according toclaim 4, wherein the polynucleotide is selected from the groupconsisting of: SEQ ID NO: 2; SEQ ID NO: 3; the DNA form or DNA/RNAhybrid forms thereof; and any complementary sequence thereof.
 6. Amethod according to claim 5, wherein the polynucleotide is operablylinked to a suitable promoter, for instance a salivary gland-specificpromoter.
 7. A method according to claim 4, wherein a plasmid comprisingthe polynucleotide is administered.
 8. A method according to claim 5,wherein a plasmid comprising the polynucleotide is administered.
 9. Amethod according to claim 6, wherein a plasmid comprising thepolynucleotide is administered.
 10. A method according to claim 7,wherein a viral vector comprising the plasmid is administered.
 11. Amethod according to claim 1, wherein the composition comprising thepeptide or analogue thereof is administered in the form of apharmaceutically acceptable composition.
 12. A method according to claim2, wherein the composition comprising the peptide or analogue thereof isadministered in the form of a pharmaceutically acceptable composition.13. A method according to claim 3, wherein the composition comprisingthe peptide or analogue thereof is administered in the form of apharmaceutically acceptable composition.
 14. A method according to claim4, wherein the polynucleotide is administered in the form of apharmaceutically acceptable composition.
 15. A method according to claim5, wherein the polynucleotide is administered in the form of apharmaceutically acceptable composition.
 16. A method according to claim6, wherein the polynucleotide is administered in the form of apharmaceutically acceptable composition.
 17. A method according to claim7, wherein the plasmid is administered in the form of a pharmaceuticallyacceptable composition.
 18. A method according to claim 10, wherein theviral vector is administered in the form of a pharmaceuticallyacceptable composition.